Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector.
We have made use of the plasmid vector pBSV-8His recently established for baculovirus-mediated expression of His-tagged proteins for the production of a truncated soluble complement regulator protein. The protein comprised the N-terminal part, i.e. short consensus repeats (SCRs) 1-4, of the B-cell membrane protein complement receptor type two (CR2; CD21) and contained the functional epitopes which mediate the binding of the complement component C3 fragments C3dg and iC3b. This recombinant protein, termed rsCR2.1-4, was furnished with a C-terminal histidine-tag for easy purification from insect cell supernatant. The yield of > 90% pure rsCR2.1-4 was 3 micrograms/ml supernatant at day eight p.i. RsCR2.1-4 was expressed as two proteins with a M(r) of 29 and 31 representing differentially glycosylated forms. Both reacted specifically with anti-CR2 mAb HB5 directed against SCRs 3-4, but not with anti-CR2 mAbs recognizing SCRs beyond SCR 4. RsCR2.1-4 was able to bind C3dg and to block binding of C3dg-coated beads to Raji cells. Used as antigen for immunization, it allowed the efficient and well-aimed generation of antisera which specifically blocked attachment of C3dg-coated beads to Raji B cells. Thus, insect cell derived rsCR2.1-4 has proved a valuable tool to study the functional domain of CR2 and its immunoregulatory capacity in B-lymphocytes.
Prodinger
W M
WM
Institut für Hygiene, University of Innsbruck, Austria. wolfgang.prodinger@uibk.ac.at
Schoch
J
J
Schwendinger
M G
MG
Hellwage
J
J
Parson
W
W
Zipfel
P F
PF
Dierich
M P
MP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics* / Q000276:immunology; D011951:Receptors, Complement / Q000235:genetics* / Q000276:immunology; D017464:Receptors, Complement 3d / Q000096:biosynthesis* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D011994:Recombinant Proteins